WTA Pipeline Results Summary

Summary

Library	Total Reads in FASTQ	Pct Reads Passing Quality Filter	Pct CellLabel UMI Aligned Uniquely
N22 WTA	113,058,879	61.55%	41.66%

Putative Cells

564

Bioproduct Type	Aligned Reads By Type	Mean Reads per Cell	Mean Molecules per Cell
mRNA	8,076,193	1,502.27	72

Graphs

Select Gene or AbSeq: For large datasets, only the most highly, widely, and variably expressed genes plus all AbSeq targets are listed. The AbSeq targets and mRNA genes are sorted by total expression (highest to lowest) separately. The bioproduct with the highest expression is selected by default.

Sequence Quality

Library	Total Reads in FASTQ	Pct Read Pair Overlap	Pct Reads Too Short	Pct Reads Low Base Quality	Pct Reads High SNF	Pct Reads Filtered Out	Total Reads After Quality Filtering
N22 WTA	113,058,879	0%	34.51%	0.08%	3.86%	38.45%	69,591,971

Library Quality

Library	Total Filtered Reads	Pct Q30 Bases in Filtered R2	Pct CellLabel UMI	Pct CellLabel UMI Aligned Uniquely	Pct Reads Useful
N22 WTA	69,591,971	80.91%	65.6%	41.66%	11.61%

Alignment Categories

Library	CellLabel UMI Reads	Annotated Transcriptome Pct	Introns Pct	Intergenic Regions Pct	Antisense Pct	Not Unique Pct	Ambiguous Pct	No Feature Pct	AbSeq Pct	Sample Tag Pct	VDJ TCR Pct	VDJ BCR Pct	Unaligned Pct
N22 WTA	45,650,407	4.9%	12.79%	15.75%	13.55%	15.48%	1%	0.03%	0%	0%	0%	0%	36.5%

Reads and Molecules

Bioproduct Type	Aligned Reads By Type	Total Raw Molecules	Total RSEC Molecules	Mean Raw Sequencing Depth	Mean RSEC Sequencing Depth	Sequencing Saturation
mRNA	8,076,193	3,299,718	3,164,204	2.45	2.55	70.13

Cells

Putative Cells

564

Basic Algorithm Cells

564

Bioproduct Type	Pct Reads from Putative Cells	Mean Reads per Cell	Median Reads per Cell	Mean Molecules per Cell	Median Molecules per Cell	Mean Bioproducts per Cell	Median Bioproducts per Cell	Total Bioproducts Detected
mRNA	10.49%	1,502.27	1,189	72	51	58.83	46	10,876

Pipeline Inputs

Pipeline version: 2.0

Pipeline Start Time: Tue Jan 02 2024 16:11:41 GMT+0000 (Greenwich Mean Time)

AbSeq Reference:

No value

Reference Files Archive:

RhapRef_Human_WTA_2023-02.tar.gz

Supplemental Reference:

No value

Targeted Reference:

No value

Reads:

N22_WTA_S1_R1_001.fastq.gz
N22_WTA_S1_R2_001.fastq.gz

Multiplexing Settings

Sample Tags Version: No value
Tag Names:

VDJ Settings

VDJ Species Version: No value

Putative Cell Calling Settings

Enable Refined Putative Cell Calling: False
Exact Cell Count: No value
Expected Cell Count: No value
Putative Cell Call: mRNA

Exclude Intronic reads: False

Generate Bam output: True

Metric Alerts

(Under development)

For the [N22_WTA] library, the [Pct_Reads_Too_Short] metric value of 34.51% is greater than 5% (Sequencing Quality section, WTA assay threshold).

May be due to short products in the library prep.
Look for evidence of this in QC traces and contact customer support for suggestions on additional cleanups.

For the [N22_WTA] library, the [Pct_CellLabel_UMI] metric value of 65.6% is less than 85% (Library Quality section, WTA V2 Bead assay threshold).

May be due to poor sequencing or library quality, or incorrect reference panel.
Check Q30 scores and QC traces, and confirm that the appropriate transcriptome reference and complete AbSeq reference was used.
This can also occur if V1 and V2 beads are combined in a single library.

For the [N22_WTA] library, the [Unaligned_Pct] metric value of 36.5% is greater than 10% (Alignment Categories section, WTA assay threshold).

May occur in experiments with low cell input. This can be addressed with additional cleanups for low cell input libraries (included in the protocol).
Can also result from low cell viability or poor sequencing or library quality.
This is expected if samples such as nuclei containing a high proportion of unprocessed transcripts are being run.

For the [mRNA] bioproduct type, the [Pct_Reads_from_Putative_Cells] metric value of 10.49% is less than 75% (Cells section, WTA assay threshold).

May be an indication of low cell viability going onto the cartridge.
Confirm on scanner that an appropriate proportion of live cells were captured.